Mobile lab: A novel pathogen assay using the nucleic acid automatic assay system assisted by a self-contained microfluidic cassette and chitosan decorating magnetic particles

Abstract

Microfluidic pathogen detection cartridges with integrated switching valves have gained significant attention. Traditionally, these microfluidic cartridges utilized magnetic beads for a “bind-wash-elute-reaction” protocol to extract DNA but often lacked PCR-based aerosol contamination validation and focused primarily on isothermal applications. There is room to improve these systems, such as using novel DNA extraction methods and integrating PCR as the gold standard. In this study, we developed chitosan-decorated magnetic (CDM) particles and incorporated them into a mass-produced, self-contained microfluidic cartridge for the first time. Our results demonstrated that the DNA can be efficiently enriched from the lysis buffer (pH 5) using 75 % degree of deacetylation and 1 % of chitosan-modified CDM particles (20 μL). The DNA extraction efficiency exceeded 90 %. The CDM particles enabled “in-situ PCR”, simplifying the on-chip detection process to a “bind-wash-reaction” protocol. Additionally, we developed a nucleic acid automatic assay system (NA3 system) featuring a fluid thermocycler, four-channel optical detection, magnet, microcontroller, and software. The entire assay, from sample preparation to detection, can be completed within approximately 60 minutes, simultaneously detecting 16 targets. The performance of the NA3 system was better than that of the pristine magnetic microparticles-based assay and the commercial magnetic beads kit-based assay. The linearity, specificity, multi-pathogen detection, and user-friendliness of the NA3 system were tested. We validated the NA3 system's performance in detecting food-borne and serum infectious pathogens, demonstrating its potential for use in resource-limited settings.